Pilot Test of a Novel Behavioral Intervention on BP Control in HTN Patients

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The mechanisms of action of vitamin E, a potent lipid soluble antioxidant that is necessary for reproduction, are unknown [1]. A dichotomy exists between vitamin E’s antioxidant and biologic activities, in that plants synthesize eight different molecules with vitamin E antioxidant activity, yet only one of these, -tocopherol, is a nutrient required by animals. We believe that the lack of success in determining the molecular function of -tocopherol results from two major obstacles. First, -tocopherol requires special transport mechanisms for delivery to tissues and is involved in complex interactions between various oxidizing and antioxidant systems; these factors make outcomes from cell culture studies largely misleading. Secondly, it is difficult using experimental animals to obtain tissues (especially embryos) that are sufficiently -tocopherol-depleted to be useful to identify -tocopherol-sensitive functions. To obviate these problems, we propose to use vitamin E-depleted zebrafish (Danio rerio) as a model system. Zebrafish are vertebrate animals with genes generally homologous to those of humans, large numbers of animals can be raised, diets can easily be manipulated, targeted genes can be readily modified, and embryonic stages can be studied over the course of development. Critically, the -tocopherol transfer protein (-TTP) is expressed in human yolk sac [2], is abundantly expressed by the 48 hour postfertilization (hpf) zebrafish embryo and its expression increases with oxidative stress [3]. Therefore, - TTP likely mediates -tocopherol transfer to the embryo from the yolk sac. To study -tocopherol-deficient (E-) zebrafish, we have developed a defined, -tocopherol-deficient diet and demonstrate in preliminary studies that E-zebrafish are fertile and capable of producing viable, Eembryos. By 48 hpf, many E- embryos exhibit severe developmental malformations. Defects include craniofacial and cardiovascular abnormalities, thereby linking -tocopherol requirements to neurological and cardiovascular development. Based on our literature review, we hypothesize that -tocopherol provides antioxidant protection for specific, key lipid mediators necessary for embryonic development, including cell loss via programmed cell death. Specifically, glutathione peroxidase 4 (GPx4), a detoxifier of phospholipid hydroperoxides, senses and translates oxidative stress into cell death that is mediated by both 12/15-lipoxygenase (12/15 LOX) and apoptosis-inducing factor (AIF) [4]. Importantly, -tocopherol, but not water-soluble antioxidants, efficiently prevented cell death. These findings support the hypothesis that -tocopherol is necessary during embryogenesis to modulate lipid mediators involved in specific developmental steps, especially in the nervous system. To test this hypothesis, we propose the following aims: Aim 1. Define the role of the -tocopherol transfer protein (-TTP) during embryonic development Hypothesis: -TTP is present in the yolk sac of the embryo, and is expressed in increasing amounts during embryonic development, to facilitate -tocopherol transfer to key sites. -Tocopherol is necessary for the earliest cells in embryonic development to allow appropriate lipid-mediator regulation. Aim 2. Define the roles of oxidant and antioxidant apoptosis regulators during embryonic development Hypothesis: -tocopherol is necessary to prevent abnormal lipid peroxidation of key mediators. 12/15- LOX increases lipid peroxidation thereby signaling AIF translocation and cell death. Opposing 12/15-LOX is GPX4, which uses glutathione (as a co-factor) and detoxifies phospholipid hydroperoxides. Thus, GPX4 suppresses lipid peroxidation. -Tocopherol not only stops the lipid peroxidation chain reaction, it is absolutely required for reproduction. Therefore, -tocopherol will prevent, and its absence will potentiate, developmental abnormalities if either GSH synthesis or GPX4 expression is abrogated. If 12/15-LOX or AIF expression is knocked down, -tocopherol will have a lesser effect. Aim 3. Define the role of -tocopherol in the developing nervous system Hypothesis: The nervous system is especially sensitive to -tocopherol deficiency; therefore, the lack of -tocopherol in the developing embryo will result in specific nervous system malformation and dysfunction. The zebrafish model is ideal for our studies because it, like humans, has a preference for -tocopherol, expresses the ttp gene and requires vitamin C, an important determinant in -tocopherol antioxidant function in humans [5]. Moreover, the -tocopherol deficient zebrafish embryo displays characteristic abnormalities suggesting dysfunctional neurological and cardiovascular development. Our long-term goal is to define molecular targets altered by -tocopherol-deficiency because this mechanistic data can then be tested in other model systems, ultimately translating these results to our basic understanding of why humans require -tocopherol, not just for reproduction, but also to maintain a healthy nervous system. The zebrafish allows us to distinguish the effect of -tocopherol deficiency in the embryo itself, separate from maternal -tocopherol deficiency during gestation. With that in mind, our approach is to let our biological system respond to the insult (-tocopherol-deficiency), and then pursue the underlying mechanism by exploiting the advantages of the zebrafish model. Using this approach, our zebrafish studies will allow us to document specific -tocopherol molecular functions, key antioxidant/oxidant signaling mechanisms, as well as determining at what stages -tocopherol is required